Pulsed laser ablation of WSe2 was carried out in H2S gas under appropriate laser fluence and reactive gas pressure. It was found that moderate sulfur doping (S/Se ~0.2–0.3) leads to significant improvement in the tribological properties of WSexSy/NP-W coatings at room temperature. Changes in the coatings during tribotesting depended on the load on the counter body. The lowest coefficient of friction (~0.02) with a high wear resistance was observed in a N2 environment at an increased load (5 N), resulting from certain structural and chemical changes in the coatings. A tribofilm with a layered atomic packing was observed in the surface layer of the coating. The incorporation of nanoparticles into the coating increased its hardness, which may have influenced the formation of the tribofilm. The initial matrix composition, which had a higher content of chalcogen atoms ((Se + S)/W~2.6–3.5), was altered in the tribofilm to a composition close to the stoichiometric one ((Se + S)/W~1.9). W nanoparticles were ground and retained under the tribofilm, which impacted the effective contact area with the counter body. Changes in the tribotesting conditions—lowering the temperature in a N2 environment—resulted in considerable deterioration of the tribological properties of these coatings. Only coating with a higher S content that was obtained at increased H2S pressure exhibited remarkable wear resistance and a low coefficient of friction, measuring 0.06, even under complicated conditions.A novel laser-based method for producing nanocomposite coatings consisting of a tungsten sulfoselenide (WSe Transition Metal Dichalcogenide (TMD) coatings are highly regarded for their exceptional solid-lubricating properties. Research into TMD coatings has been ongoing for several decades, with a growing emphasis on exploring their properties ,2,3,4,5.The most widely utilized method for obtaining TMD-based coatings is ion sputtering, also known as magnetron deposition. This technique allows the composition of deposited coatings to be flexibly adjusted and the tribomechanical characteristics to be improved. This is achieved through forming multilayer or compound coatings with metals  to enhance their corrosion resistance and low-friction properties via intercalation ,17,18. I2 and MoSe2 targets produces b.c.c. Mo nanoparticles [2 targets caused β-W nanoparticles (NP-W) to be deposited on the substrate surface [Pulsed laser deposition (PLD) is used more rarely in producing TMD-based coatings than magnetron deposition. Although initial experiments and subsequent studies have demonstrated the potential of PLD in obtaining TMD coatings, it remains limited due to the complexity of laser systems and the challenges in depositing coatings on larger surfaces. Yet, the unique features of PLD, such as versatility, ease of integration with ion irradiation, and the ability to control ion-plasma flux energy, offer new avenues for exploring the development of TMD coatings with improved characteristics ,35,36,37articles . Meanwhi surface . In the  surface . Note th surface ,42,44. T surface .2-based coatings with an increased concentration of W nanoparticles were obtained, and their chemical state and structure were analyzed. The chemical composition of the coatings was modified by introducing sulfur atoms. Several previous studies have established for several coatings produced by magnetron and laser deposition that mixing different chalcogens (S and Se) has a positive effect on the tribological properties of TMD coatings [x/NP-W coatings with sulfur. WSexSy/NP-W coatings have been formed by pulsed laser ablation of the WSe2 target in hydrogen sulfide at different pressures. The tribological properties of WSexSy/NP-W coatings with different S concentrations were investigated in wet air and a N2-enriched environment at room temperature. Additionally, this study presents the results of tribotests conducted at −100 °C in a N2-enriched environment, which demonstrated the superior performance of WSexSy/NP-W coatings with a high concentration of chalcogen atoms under these complicated sliding conditions.If the density of the metal nanoparticles is high, they can have a significant impact on the tribological properties of TMD-based coatings. On the one hand, these particles can cause accelerated abrasion of the coating; on the other hand, rounded particles can affect contact with the counter body. The contact area may change, or sliding friction may turn into rolling friction. To investigate this issue, in this study, WSecoatings ,17,35,462 target for laser ablation was synthesized using a cold-pressing technique with WSe2 micropowder of 99.9% purity. The details of the target synthesis are described in a previous publication [2, with a pulse repetition rate of 20 Hz. The laser beam was directed at a 45-degree angle to the surface of the target, and an optical scanning system was used to move the beam across the surface. The silicon substrates, which were selected based on the favorable tribological properties of WSex coatings on Si-containing underlayers, found by Shtansky et al. [−3 Pa. To alloy the WSex/NP-W coatings with sulfur, hydrogen sulfide gas was introduced into the chamber at pressures of 3.6 and 9 Pa. The obtained coatings were designated as WSexSy/NP-W_3.6 and WSexSy/NP-W_9, respectively. The substrate temperature was maintained at room temperature, and the pressure was selected based on prior studies on TMD film deposition by RPLD [The WSelication . The tary et al. , were po by RPLD . The dep7/2 core-level lines located at a binding energy of 84.0 eV. The structure of the coatings before and after tribotesting was studied by micro-Raman spectroscopy (MRS) using a 632.8 nm (He–Ne) laser; the laser-beam cross-section was less than 1 μm. The modes of MRS spectrum measurements (laser-beam intensity and measurement time) were selected in such a way that structural and chemical changes occured in the coatings.The surface morphologies of the fabricated coatings and their chemical composition were studied by scanning electron microscopy and by energy-dispersive X-ray spectroscopy . The crystal structure of the coatings was examined by grazing incidence X-ray diffraction (XRD) using an angle of 10° and Cu Ka radiation in an Ultima IV  diffractometer. The chemical states of the coatings were studied by X-ray photoelectron spectroscopy (XPS). The XPS spectra were obtained by a Theta Probe Thermo Fisher Scientific  spectrometer with a monochromatic Al Kα X-ray source (1486.7 eV) and an X-ray spot size of 400 μm. The photoelectrons had a take-off angle of 50° with respect to the surface plane. The spectrometer energy scale was calibrated using Au4fxSy/NP-W-coated silicon substrate was prepared using a focused ion beam and investigated by STEM with an accelerating voltage of 200 kV. The protective Pt/C film was applied before sample preparation for STEM studies. The acquired images were processed with the help of Digital Micrograph software  and TIA software (Thermo Fisher Scientific).The structure and composition of the coatings at the nanoscale were analyzed before and after tribological testing using scanning transmission electron microscopy (STEM) and energy dispersive spectroscopy (EDS) on an OSIRIS TEM/STEM instrument  equipped with a Bruker EDS detector. A thin cross-section of the WSe2-enriched environment (RH~7%). In some cases, the tested Si plate was mounted in a holder cooled with liquid nitrogen. To prevent water vapor condensation and ice layer formation, the friction pair was treated in a N2-enriched environment. An optimal N2 flow rate made it possible to keep the plate temperature at about −100 °C and remove the air from the area surrounding the tested sample. The characteristics of the wear tracks on the coatings, the wear scar on the balls, and the wear debris were obtained using an optical profilometer and optical microscopy. After tribotesting, additional studies of the wear tracks by STEM, EDS, and MRS were carried out.The indentation hardness of the films was determined using a Nano Hardness Tester  equipped with a Berkovich diamond indenter tip, calibrated against fused silica. The tribotesting of the thin-film coatings was carried out by an Anton Paar TRB3 tribometer in a reciprocating motion mode, using a steel ball (100Cr6) with a diameter of 6 mm as a counter body. The loads were 1 and 5 N. The sliding speed of the ball on the coated substrate was 2 cm/s. The tribotests were performed at 22 °C in air at a relative humidity of RH~35% and in a Nx/NP-W and WSexSy/NP-W coatings deposited on a substrate during 25 min. The coatings have a sufficiently dense structure, which is weakly dependent on the deposition conditions. The surface showed submicron rounded particles consisting of a cluster of smaller spherical nanoparticles. On the surface of the WSex/NP-W coating, there was only a small number of spherical particles, with diameters of up to 0.5 µm.2 target subjected to pulsed laser ablation revealed the formation of rounded particles enriched in tungsten. The SEM, EDS, and XRD results of the laser-irradiated WSe2 target are shown in 3O and β-W. The stabilization of the β-W phase is attributed to the introduction of O  atoms. The analysis of peak intensities suggests that the formation of α-W was predominant on the surface of the target.The study of the WSe2 target requires further investigation. This mechanism encompasses the formation of superheated liquid W-Se phase, potentially accompanied by the selective evaporation of selenium and the coagulation of the thin film of W-enriched liquid into spherical particles that then solidify. When the laser impacts a local target area, the particles are likely to be captured by the laser plume and transported to the coating surface. Large particles have low velocities and are unlikely to adhere to the coating. Smaller particles (nanoparticles) exhibit high velocities (~2 × 104 cm/s) and are more likely to adhere to the coating surface [The mechanism behind the modification of phase composition in a laser-ablated WSe surface .x/NP-W coating confirmed the formation of a structure consisting of an X-ray amorphous matrix and a β-W/W3O phase /W ratio was 2.4. An increase in the H2S pressure to 9 Pa led to a further rise in sulfur content, resulting in a S/Se ratio of 2.3 and a (Se + S)/W ratio of 4.3. According to EDS (SEM) measurements, the oxygen concentration in WSex/NP-W and WSexSy/NP-W coatings was ~3–4 at.%. Considering that the particles in these coatings contain mainly tungsten, the amorphous matrix of these coatings would be significantly enriched in chalcogen atoms.EDS (SEM) elemental analysis showed that in a relatively large volume of WSexSy/NP-W coating prepared at a 3.6 Pa pressure of reactive H2S gas for 15 min. The coating had a thickness of approximately 900 nm. The high-angle annular dark-field images and elemental maps reveal that the WSexSy/NP-W coating was composed of a Se- and S-enriched matrix with embedded W-enriched nanoparticles. The coating contained regular, spherical particles with a diameter of up to 100 nm, in addition to particles of a deformed ellipsoid shape. The latter particles had a size of less than 50 nm. The density of the nanoparticles was high, while the coating exhibited a relatively dense structure with pores of up to 50 nm. Observations showed that the height of the surface protrusions on the coating did not exceed 100 nm. The analysis of high-resolution TEM (HRTEM) images and selected area electron diffraction (SAED) patterns confirmed the formation of nanoparticles with the characteristic crystal structure of the β-W phase /W ratio of approximately 2.6–3.5. The oxygen concentration did not exceed 6 at.%. A cross-sectional analysis of lager areas that included the NP-W showed a (Se + S)/W ratio of approximately 1.7, suggesting that the W atom content in the matrix and the NP-W was approximately equal. However, the EDS analysis of the nanoparticles indicated that they may also contain Se, S, and O atoms—possibly fragments of the matrix enriched with chalcogen atoms. With an estimated specific density of the amorphous matrix being 2–3 times lower than that of tungsten, the volume fraction of nanoparticles in the coating is estimated at 25–30%. The EDS elemental analysis of nanoparticles showed that they could contain Se, S, and O atoms, in addition to tungsten. Nevertheless, these results are not sufficiently reliable, as fragments of the matrix enriched with chalcogen atoms may have entered the measurement region.x/NP-W and WSexSy/NP-W coatings. The spectrum of WSex/NP-W coatings is in many ways like that obtained earlier for WSex-based coatings deposited by PLD and magnetron techniques [−1, which is characteristic of an amorphous/disordered local structure. The introduction of sulfur reduced the intensity of this broad peak, indicating an increased degree of local packing defectiveness in WSexSy/NP-W films. The obtained films are in a highly nonequilibrium state, which can be easily altered by mild heating using a high-intensity laser beam. Upon laser heating, a narrower peak at approximately 250 cm−1 emerges (as shown in 1 2g and A1g) from the WSe2 compound. Nevertheless, peaks related to various Se molecular configurations are also present in this frequency range [x/NP-W coating, surplus selenium may be distributed along the interstitials of the 2H-WSe2 lattice. Other changes, such as the formation of elemental Se inclusions or sublimation/evaporation of excess selenium, are also possible.chniques ,49. All cy range . Thus, dx/NP-W and WSexSy/NP-W coatings. The analysis of the W4f spectra reveals the presence of W0, W4+, and W6+ species [4+ and W6+ states is due to tungsten’s chemical bonds with chalcogen atoms (WSe2/WS2) and oxygen (WO3), respectively. An increase in sulfur concentration results in a shift of the W4f doublet peaks. The peaks for W4f7/2 and W4f5/2 in the WSex/NP-W coating had binding energies of 32.16 and 34.23 eV, respectively. For the WSexSy/NP-W_9 coating, these peaks were located at slightly higher binding energies of 32.47 and 34.52 eV. This energy shift and its increase with growing S concentration is a typical feature observed in metal diselenides upon partial replacement of Se atoms with S atoms [0 to the total spectrum indicated that the W nanoparticles were effectively enveloped by the matrix material, even on the surface. It is possible that the W nanoparticles located on the coating surface oxidized, leading to relatively intense peaks in the XPS spectra due to W6+. The binding energies of the W4f7/2 and W4f5/2 peaks caused by metal oxide inclusions were almost independent of the coating composition and were found to be approximately 35.7 and 37.8 eV, respectively. species ,53,54. T S atoms ,35,55. TxSy/NP-W coatings reveal the presence of S2p peaks, along with Se3p peaks in the background. The Se3p peaks were analyzed using information obtained from the Se3p spectra of the pure WSex/NP-W coating. The two peaks at 161.8 and 163.0 eV shown in the XPS spectra of the WSexSy/NP-W coatings can be assigned to S2p3/2 and S2p1/2 binding energies, respectively [3/2 and Se2p1/2 were observed at 160.9 and 166.6 eV, respectively.The XPS spectra of the WSeectively ,35. The x/NP-W coating, the overstoichiometric composition of the matrix was considered. Two pairs of peaks were necessary to fit each Se peak, corresponding to Se2− species located at different nodes in the amorphous mesh of the WSex matrix and to Se0 species in Se-enriched clusters [When analyzing the Se3d region in the XPS spectrum of pure WSeclusters ,49,54,565/2 peak for these Se species are 54.4 and 55.3 eV, respectively. The S incorporation in the WSexSy matrix slightly increases the binding energy for the Se2− peak and introduces a broad band at higher binding energies (over 57 eV), which could be due to the introduction of O and/or S atoms into the Se clusters. Surface oxidation of the coatings caused by prolonged exposure to air after coating deposition may also influence the chemical state of tungsten and selenium. The best fit of the experimental Se3d spectrum was obtained when the Se3d5/2 peak was at 57.8 eV, which corresponds to Se2+ species. Se-enriched clusters may form due to excess Se atoms segregating. During the formation of the WSexSy/NP-W coatings by reactive PLD, Se and S atoms were embedded into the coating matrix through different mechanisms. Se atoms were introduced through physical vapor-phase deposition, while S atoms were probably introduced through chemical interaction with surface coating atoms. High chalcogen concentrations in the WSexSy matrix may result in Se phase segregation, as observed in previous studies of PLD processes in Se-based TMD films [The binding energies of the Se3dMD films ,56.x/NP-W and WSexSy/NP-W coatings at room temperature in environments consisting of moist air and a N2-enriched environment under loads of 1 and 5 N. The coating deposition time was 25 min. For the WSex/NP-W coating, measurements in the air under a load of 1 N exhibited a behavior characterized by an initially higher coefficient of friction (CoF~0.09), which transitioned (run-in) to a lower steady-state CoF~0.07 with increased sliding cycles. This run-in behavior changed when this coating was doped with sulfur. For the WSexSy/NP-W_3.6 coating, the initial CoF value was 0.05. During the run-in phase, the CoF increased to 0.06 and then slowly increased to 0.07 by the end of the test. For the WSexSy/NP-W_9 coating, the CoF increased relatively quickly from an initial value of 0.06 to 0.12 when tested for 1000 cycles. The testing of this coating was then stopped.2-enriched environment with loads of 1 N showed only a slight improvement in the friction properties of the WSex/NP-W coating, particularly during the running-in phase, where no increased coefficient of friction was observed. Throughout the tribotesting, the steady-state CoF was ~0.05–0.06. In contrast, the addition of sulfur considerably improved the low-friction properties of the WSexSy/NP-W_3.6 coating in this environment, resulting in a steady-state CoF of ~0.045. Changing the test environment did not improve the tribological properties of the WSexSy/NP-W_9 coating.The tribotests in a Nx/NP-W and WSexSy/NP-W coatings when tested in a N2-enriched environment. For WSex/NP-W, the CoF value decreased to 0.03–0.04, but a sharp increase in CoF was observed at a certain stage of the tribotest. The durability of the coating did not exceed ~1500–2000 sliding cycles. This behavior was repeated in three tribotests. The best tribological properties were found for WSexSy/NP-W_3.6, with a CoF value of 0.02–0.025 throughout the entire tribotest period. Increasing the load caused some improvement in the tribological properties of the WSexSy/NP-W_9 coating. During a sufficiently long sliding period (up to 2500 cycles), the CoF value did not exceed 0.06.Increasing the load on the counter body had a different effect on the tribological behavior of the WSexSy/NP-W_9 coating indicate that coatings with a high concentration of chalcogen atoms had unsatisfactory tribological properties . You. Youx/NPd 60 GPa ,49. The x/NP-W and WSexSy/NP-W coatings have revealed that a comparison of the wear behavior of WSex/NP-W and WSexSy/NP-W_3.6 coatings is of particular interest under conventional test conditions (as regards ambient temperature and environment). The wear investigation focused on the central area of the track, as there was no significant correlation between wear and ball sliding speed in different areas of the track. Under specific test conditions, such as low temperatures and an environment containing water vapor, the sliding properties of these coatings were considerably impaired compared with the WSexSy/NP-W_9 coating. Additional investigations are needed to determine the underlying reasons for this behavior. The Studies into the tribological properties of WSex/NP-W and WSexSy/NP-W_3.6 coatings and their corresponding counter bodies after tribotests in air. The coatings were deposited for 25 min. Notably, a correlation between the frictional and wear behavior of these coatings can be observed. The WSexSy/NP-W_3.6 coatings and counter bodies experienced slightly higher wear than their WSexSy/NP-W counterparts, resulting in a wider and deeper wear track and increased counter-body wear scar size. The depth profile of the wear track on the WSexSy/NP-W_3.6 coating differed slightly from that of the WSex/NP-W coating, with the former exhibiting large areas where the coating was completely removed from the substrate. The sharp edges of these areas suggest that the WSexSy/NP-W_3.6 coating may have peeled off in relatively large fragments, whereas track deepening due to wear and tear on the coating was less prominent.xSy/NP-W_3.6 coating to the substrate compared with the WSex/NP-W coating could be attributed to the decreased energy of deposited atoms and ions resulting from deposition in a reaction gas. This decrease in energy reduces the efficiency of chemical bond formation at the coating–substrate interface, and in turn, the efficiency of surface sputtering [x/NP-W coating measuring 800 nm and the WSexSy/NP-W_3.6 coating 1200 nm.The weaker adhesion of the WSeuttering . As a rex/NP-W and WSexSy/NP-W_3.6 coatings and counter bodies after the tribotests in a N2-enriched environment under a load of 1 N. The WSexSy/NP-W_3.6 coating showed clearly improved wear resistance compared with the WSex/NP-W coating. At the same time, the wear of the counter body was significantly reduced. The addition of S caused a reduction in track width from 150 µm to 80 µm. The maximum track depth reduced from 240 to 30 nm by a factor of 8. A comparison of the wear of these coatings in wet air and nitrogen showed that in N2 environment the wear decreased for both coatings. This behavior is typical of all TMD-based coatings. However, for WSexSy/NP-W_3.6 coating, this reduction was the most significant.x/NP-W coating in N2-enriched environment with an increased counter body load of 5 N (Hertzian contact stress ~0.86 GPa), a marked increase in wear was observed. xSy/NP-W_3.6 coating, the increment was moderate. The average depth of the track rose to 40 nm, with some regions of the track exhibiting a wear depth increase to 100 nm. The width of the well also increased from 80 nm to 140 nm, with a concomitant growth in the wear scar of the ball. Notably, the exposure of the counter body to a high load resulted in the delamination of the coatings from the substrate in some localized areas of the track, as evidenced by the formation of visible swellings in the microphotographs of the tracks 2 nanoclusters cannot be ruled out.The WSeanoforms . In cont lattice . The asy−1. The broad band extending from 600 to 800 cm−1 is situated in the region of W–O–W stretching bonds of WO3 [−1 indicated a high degree of WO3-phase defectiveness and points to the possible formation of Fe-containing oxides  [2-enriched environment reduced the influence of oxygen and water vapor on tribo-chemical processes in the friction zone but did not eliminate it. This phenomenon is more pronounced for the S-containing WSexSy/NP-W coating as compared with pure WSex/NP-W coating.Raman studies of wear debris showed that, in addition to the S/Se-containing phase, there was a metal oxide phase as well a,b. The s of WO3 ,59. The xSy/NP-W coating in the central region of the wear track formed after 500 sliding cycles in a humid air environment are presented in 2-like structure. The dimensions of the ordered areas with layered packing of atoms reached up to 20 nm, and their orientation was mainly influenced by the nearest W nanoparticle. The predominant orientation of the basic planes was along the surface of the nanoparticle, potentially due to the movement or vibration of the nanoparticle within the amorphous matrix. The interplanar spacing varied between 0.65 to 0.7 nm. No evidence of tribofilm formation with WSe2 nanocrystals oriented along the surface was found, nor was any change in the Se/S and (Se + S)/W ratios in the near-surface layer of the matrix detected. Exposure to moist air resulted in an increase in the O atoms concentration in the matrix surface layer to 11.7 at.%, with O atoms penetrating to a depth of 30 nm.Results of the STEM analysis of the WSexSy/NP-W_3.6 coating and the effective formation of a W(Se/S)2-based tribofilm on the surface. 2-enriched environment after 1000 sliding cycles. The thickness of the tribofilm possessing ordered W(Se/S)2 fringes reached 15 nm, and the basic planes had a preferential orientation parallel to the coating surface. The distance between the basic planes was approximately 6.7 nm. At the boundary between the film and the matrix, the atomic planes exhibited arbitrary orientations. The thickness of such a transition layer did not exceed 10 nm. In deeper layers, the amorphous matrix structure was mostly preserved. The tribo-induced modification of the surface-layer structure involved the partial removal of S and Se atoms, resulting in a decrease in the (Se + S)/W ratio to ~1.9. However, the O atoms’ concentration did not change significantly in the surface layer after tribotesting.Increasing the counter-body load to 5 N resulted in noticeable compaction of the WSe2 can cause the intercalation effects that increase the interplanar distance. This is evident from the increase in the base-plane spacing to 0.7 nm, as shown in xSy/NP-W_3.3 coating in a N2-enriched environment at a load of 1N occurred in a similar way, the matrix crystallization throughout the coating can provide sufficient low-friction properties, even at a high concentration of chalcogen atoms—(Se + S)/W ~2.6–3.8. However, the results of tests under a higher load show that the densification of the structure and the formation of a tribofilm with a composition close to stoichiometric by the (Se + S)/W parameter are more important. The mechanisms responsible for removing excess S and Se atoms require further investigation. These atoms may be removed through the formation of volatile compounds with oxygen and hydrogen present in the applied media, such as wet air and a N2-enriched environment with an RH~7 %.Raising the load to 5 N did not lead to an effective crystallization of the matrix in the coating volume, as was observed in the lower load tests. This lack of structural transformation could be due to the strong compaction of the coating induced by a higher load, which may have inhibited the reduction in local atomic packing density required for the transformation of an amorphous structure into a layered one. However, the transformation of an amorphous matrix with an excessive content of chalcogen atoms ((Se + S)/W ~2.6–3.8) into crystalline 2H-W(Se/S)xSy/NP-W_3.3 coating revealed that these nanoparticles did not migrate into the coating and were removed during wear with the matrix material, under both high and low loads. This was confirmed by the consistent (Se + S)/W ~1.7 values measured by EDS in the cross-section of the coating. Yet, under lower loads, the nanoparticles were found to protrude above the coating surface, resulting in increased friction /W < 3.5, sulfur atoms were dispersed throughout the matrix volume, potentially taking different states within the amorphous matrix packing. As a result, the hardness of the coating decreased, albeit the process being mitigated by the presence of W nanoparticles, promoting plastic deformation and densification of the WSexSy/NP-W_3.6 coating under load. The introduction of S atoms appeared to alter the local atomic packing and may have contributed to the transformation of the amorphous structure into a crystalline/layered structure under tribo-impact. The partial substitution of Se atoms by S atoms in the layered structure is believed to have reduced the shear stresses between the basal planes in the 2H-W(Se/S)2 lattice. Notably, Hu et al. [xSy/NP-W coatings under extremely challenging environmental conditions, such as very low temperatures and low humidity, the S content may need to be significantly increased, resulting in a much softer matrix.A comparison of the tribological properties of WSeu et al.  have shox/NP-W and WSexSy/NP-W nanocomposite coatings. At relatively low counter-body loads, the nanoparticles in the coating could be removed from the surface without fracturing. Under higher loads, the particles underwent wear. However, the formation of a W(Se/S)2 tribofilm between the steel counter body and the W nanoparticle prevented direct contact between the metals, leading to a reduced friction coefficient. Notably, the contact between the counter body and the nanocomposite coating may have been localized primarily on the W nanoparticles, thus reducing the actual contact area and ultimately contributing to a lower friction coefficient.W nanoparticles formed the hard “skeleton” of the WSex coatings synthesized by means other than PLD techniques reveals that the most comparable tribotesting conditions were achieved by Dominguez-Meister et al. [x coating deposition. Reciprocating tests were conducted at 2 N of applied load (a maximum contact pressure of ~0.83 GPa), a stroke length of 2 mm, and a 2 mm s−1 of linear speed during 2500 cycles. The tribological behavior of pure WSex remains almost unaltered, showing a similar friction coefficient (0.04–0.07) and wear rates (1.5–3 × 10−7 mm3 N−1m−1), independent of the nature of the environment (in ambient air and in dry nitrogen). Mixing WSex with carbon or MoS2 did not enhance the low-friction properties [xSy/NP-W_3.6 coatings fabricated in this study by reactive PLD showed improved low-friction properties with a minimum friction coefficient of ~0.02, compared with the already known magnetron-deposited WSex coatings. The wear resistance of the WSexSy/NP-W_3.6 coatings was not inferior to that of magnetron-deposited WSex coatings. The deposition of WSex coatings by magnetron sputtering has been extensively explored and optimized. However, the tribological performance of nanocomposite WSexSy/NP-W coatings may be further improved by optimizing reactive PLD parameters.A review of the literature on the tribological properties of WSer et al. ,49,61, woperties ,62. Howe2 target to pulsed laser ablation under vacuum conditions produces a flux of W and Se atoms, along with a flux of W nanoparticles. The maximum size of the nanoparticles can reach several tens of nanometers, although the structure of the coating is mostly made up of round-shaped particles smaller than 20 nm. The volume fraction of nanoparticles in the coatings can reach 25–30%. In the presence of H2S gas during laser ablation of WSe2 target, S atoms are introduced into the amorphous matrix of the coatings along with W and Se atoms, and their concentration increases with increasing H2S pressure. The incorporation of NP-W leads to an increased hardness of the nanocomposite coatings and may impact the contact area between the counter body and the coating.This study has shown that subjecting the WSexSy/NP-W_3.6 coatings exhibit high-level tribological properties when tested in ambient air and N2-enriched environments at room temperature. The nature of tribo-induced changes in these coatings is contingent upon the load on the counter body. At higher counter-body loads, the coatings exhibited the lowest coefficient of friction (0.02) and the highest wear resistance (~1.6×10−7 mm3 N−1 m−1) when sliding in nitrogen. During sliding, an up to 20 nm-thick tribofilm forms on the coating surface containing W(Se/S)2 nanocrystals, whose basal planes align parallel to the coating surface. Importantly, the tribofilm effectively reduces the concentration of Se and S atoms to a value near stoichiometric ((Se + S)/W~1.9) and prevents W nanoparticles from making clean contact with the steel counter body. The tribological performance of these coatings, however, noticeably declines under more challenging conditions, such as low temperature (−100 °C) and N2-enriched environments with a relative humidity of ~7%.Under moderate S atom concentration, WSexSy/NP-W_9 coatings has an adverse effect on their tribological properties under “conventional” ambient conditions. However, these coatings demonstrate the best tribological performance when tested at very low temperatures (about −100 °C), with a low wear rate and a coefficient of friction of 0.06 over 3000 sliding cycles. Other coatings experience a sharp increase in the coefficient of friction to 0.1 after just 1000 sliding cycles.Notably, the excessive S content in WSe


Ultra-short-term heart rate variability (HRV) has been validated in the resting state, but its validity during exercise is unclear. This study aimed to examine the validity in ultra-short-term HRV during exercise considering the different exercise intensities. HRVs of twenty-nine healthy adults were measured during incremental cycle exercise tests. HRV parameters  corresponding to each of the 20% (low), 50% (moderate), and 80% (high) peak oxygen uptakes were compared between the different time segments of HRV analysis (180 s (sec) segment vs. 30, 60, 90, and 120-sec segments). Overall, the differences (bias) between ultra-short-term HRVs increased as the time segment became shorter. In moderate- and high-intensity exercises, the differences in ultra-short-term HRV were more significant than in low intensity exercise. Thus, we discovered that the validity of ultra-short-term HRV differed with the duration of the time segment and exercise intensities. However, the ultra-short-term HRV is feasible in the cycling exercise, and we determined some optimal time duration for HRV analysis for across exercise intensities during the incremental cycling exercise. Heart rate variability (HRV) is the fluctuation of the instantaneous heartbeat over time . HRV is There are several methods of analysis to quantify HRV. The time- and frequency- domain are traditional HRV analysis method ,12,13. TThe recording duration used to calculate HRV parameters is one of the crucial factors in accurately quantifying HRV. A recording duration of ≥5 min has been recommended to standardize HRV recordings across different studies . On the Recently, there have been many attempts to detect metabolic changes using HRV ,11,37. IThe present study aims to propose a valid time duration for HRV analysis in exercise conditions. In the following three steps, we compared five ultra-short-term segments . First, in each of the five segments, we confirmed the changes in mean values of HRV with increasing exercise intensity while comparing physiological parameter changes. Secondly, at three levels of exercise intensities , the 180-sec segment was used as the reference duration to confirm the correlation coefficient with other time segments. Finally, we determined the differences between 180-sec and other time segments at each exercise intensity. The novelty of the present study was to reveal the valid time duration for HRV analysis across all the exercise intensities. This finding would contribute to accurate HRV analysis in non-static conditions, such as exercise.In this report, twenty-nine healthy adults were included, and their data .On the day before the exercise test, all participants were asked to avoid strenuous exercise, alcohol consumption, and caffeine consumption. In addition, the participants were required to fast for 2–3 h before the exercise test; however, they were allowed to drink water. Following the arrival of participants in our laboratory, blood samples were obtained from their cubital vein using a venous catheter, after which they rested for more than 30 min. This study adopted the ramp incremental exercise test using a cycle ergometer . The participants pedaled at 10 watts for 4 min as a warm-up, followed by a gradual increase in the exercise workload  until they were exhausted. Participants were instructed to maintain pedaling frequency at 60 revolutions per min. Exhaustion was determined as the point at which the pedaling rate fell below 50 revolutions per min. The temperature and humidity of the rooms in which the exercise test was performed were 23–24 °C and 30–60%, respectively.2 of the last 30–60 sec prior to exhaustion.The respiratory gases were measured during the incremental exercise test using a mass spectrometer  via the mixing chamber method . At the Before the exercise test, the blood lactate level was measured in a seated, resting position . Additionally, the measurement was performed at the end of every 30 sec period or every minute-long period during exercise until the blood lactate level reached 4 mmol/L. Finally, the blood lactate measurement was performed 3 min after the end of the exercise test.Intravenous blood samples to assess catecholamine levels were continuously collected from the catheter every minute during the exercise test. After that, the collected samples were centrifuged at 3500 rpm for 15 min at 4 °C and stored at −80 °C until the analysis. Plasma catecholamine concentrations were analyzed by two commercial blood analysis companies .R–R intervals during the incremental exercise test were measured using a 3-lead electrocardiogram system  and the Reflex-Meijin version 4.12 software program . Only one participant’s measurements were performed using another 3-lead electrocardiogram system  because of a system malfunction. The frequency analysis was conducted using the Memcalc/Win version 2 software program . Before the frequency analysis, the artifacts and abnormal beats were excluded using that software program according to the following criteria: (1) if the heart rate was below 30 bpm and above 220 bpm, (2) if outliers were detected by comparing adjacent R–R data, (3) if the validity was not confirmed among the successive three R–R data. After that, the power spectral densities were calculated using the maximum entropy method and the non-linear least squares method ,41. The In this report, the length of time segment to analyze HRV was set at 30, 60, 90, 120, or 180 sec. Additionally, to examine the difference in exercise intensity, we selected exercise intensities corresponding to three levels of peak oxygen uptakes  and at 80% n = 20); adrenaline and noradrenaline levels at 20% n = 24), at 50% n = 23) and 80% n = 20). There were also missing data in HRV. The frequency-domain indices of HRV corresponding to 20% n = 28). In addition, electrocardiogram data obtained using the AC-301A system was incompatible with the Kubios HRV version 4.0 software. Thus, the data pertaining to the time-domain and non-linear HRV parameters in one participant were missing (n = 28). Moreover, the DAF α1 and α2 were unable to analyze in the 30-sec segment at 20% n = 0), at 50% n = 4), and 80% n = 26), and in the 60-sec segment at 20% n = 26). The SampEn was also unable to analyze in the 30-sec segment at 20% n = 0), at 50% n = 7), and 80% n = 27).There were missing data related to physiological parameters as follows: blood lactate at 50% r) or Spearman’s rank correlation coefficient (ρ). In this study, the criterion for a high correlation coefficient was set at ≥0.8. Then, the Bland-Altman analysis was conducted to confirm bias (the mean difference) and 95% limits of agreement . Finally, the bias between two different ultra-short-term HRVs was quantified using Cohen’s d statistics. For the variables with skewed data distributions, the effect size was calculated using a formula as follows:Descriptive data are presented as the mean ± SD. The normality of the data distribution was confirmed using the Shapiro–Wilk test. Based on previous recommendations , HRV datp-values was set at less than 0.05. Statistical analyses were performed using the SPSS software ver. 28 . The bias and 95% LOA calculations were conducted using the GraphPad Prism version 9.2.0 software .The Z-value was calculated using the Wilcoxon signed-rank test. In this study, the criterion for small effect size was set at ≤0.5 . The num2, tidal volume, respiratory rate, blood lactate, and noradrenaline levels increased from 20% to 50% p < 0.05). Log LF/HF in the 180-sec segment did not differ significantly among the three exercise intensities. However, log LF/HF in the 120-sec, 90-sec, and 60-sec segments decreased at 80% p < 0.05). Log LF/HF in the 30-sec segment decreased at 80% p < 0.05). Log Heart rate/LF increased at 50% p < 0.05). Among the non-linear HRV parameters, DFA α1 in the 180-sec, 120-sec, and 90-sec segments increased from 20% p < 0.05). DFAα1 decreased in the 60-sec segment at 80% p < 0.05). SampEn decreased at 50% p < 0.05). In contrast to DFA α1, DFA α2 increased with increasing exercise intensity in all ultra-short-term cases (p < 0.05). Due to missing data, the 30-sec segment of DFA α1, α2 and SampEn were not statistically analyzed.p < 0.05) except for log SDNN between the 180-sec and 30-sec segments at 80% For the time-domain HRV parameters (log SDNN and log RMSSD), the correlation between the 180-sec segment and other ultra-short-term segments were high at all exercise intensities . The log HF component was indicated to be highly correlated between the 180-sec and 120-sec, 90-sec, and 60-sec segments at all exercise intensities . In terms of the log LF/HF ratio, the correlation between 180-sec and 120-sec segments was high at 20% and 80% p < 0.05) but not at 50% p < 0.05); however, its value at 50% and 80% p < 0.05).For frequency components, the log LF component exhibited highly correlation between the 180-sec and 120-sec, 90-sec segments at all exercise intensities , but it was below the criterion at 50% p < 0.05). SampEn exhibited a high correlation between the 180-sec and 120-sec segment at 20% and 50% p < 0.05). The 30-sec segment’s DFAα1, DFAα2 and SampEn at 20% In non-linear parameters, the DFAα1 demonstrated a high correlation between the 180-sec and, 120-sec, 90-sec segments at 20% and 80% ing data .Based on these results, the acceptable minimum time segment of HRV analysis according to the correlation coefficient were summarized in In addition, the bias between two different ultra-short-term HRV parameters was quantified by the effect size . The BlaFor the log SDNN and log RMSSD values in the time-domain analysis, the bias and LOA at 20% In the log LF, HF and Heart rate/LF also, the bias and LOA at 20% 2peak, but the biases at 80% Regarding DFAα1, the bias in all ultra-short-term segments was small at 20% , and 50% VOBased on these results, the acceptable minimum time segment of HRV analysis according to the effect size were summarized in In this study, we examined the validity of ultra-short-term HRV during exercise with respect to exercise intensity. Consequently, although the ultra-short-term segments of mean HRV indicated the similar changes in HRV parameters with increasing exercise intensity, we found differences in the validity based on the duration of HRV analysis time segment and the level of exercise intensity.The changes in mean HRV parameters during exercise were almost consistent with the previous studies. The time-domain (SDNN and RMSSD) and frequency-domain indices (LF and HF components) have been reported to decrease during exercise in many previous studies ,47,48,49The mean HRV values indicated similar changes during exercise in all ultra-short-term segments. Still, the trends seem to be not necessarily matched between the 180-sec segment and other ultra-short-term segments. The previous study has demonstrated similar results in stress test (a head-up tilt) . In partThe differences (bias) between ultra-short-term HRVs increased as the time segment became shorter. It was particularly conspicuous at low exercise intensity compared to moderate to high exercise intensity. It is simply considered that the difference in the amount of heartbeat data could affect HRV analysis. The HRV is higher in lower exercise intensity, and it non-linearly decreases with an increase in HR ,57. TherRecently, HRV assessment has become more efficiently conducted with the spread of wearable devices. The previous study for athletes has been suggested to measure the ultra-short-term HRV before training in multiple days for monitoring condition and training adaptation . AccordiThere are several limitations to the present study. First, the present study adopted an incremental exercise test with the ramp method. Therefore, the validity of ultra-short-term HRV during steady-state exercise was unclear. The steady-state exercise might obtain stable HRV data that excluded the effect of non-linear changes observed in the ramp method. In addition, it was also unclear whether there was a difference in the 300 sec (5-min) segment. The time segment length in HRV measurement and analysis is recommended at 300 sec . HoweverThe validity of ultra-short-term HRV was different for exercise intensities. However, the ultra-short-term HRV is feasible in the cycling exercise, and we determined some optimal time duration for HRV analysis for across exercise intensities during the incremental cycling exercise in particular.


Nature Cell Biology 10.1038/s41556-022-01082-9. Published online 2 February 2023.Correction to: In the version of this article initially published, Supplementary Videos 1–8 were linked out of order, which caused Supplementary Video 1 to be inaccessible. The Supplementary Videos are now appropriately linked in the HTML version of the article.


G-SPI exhibits the highest disulfide bond content and the best gel properties. Compared with MP gel, the addition of H-SPI, A-SPI, G-SPI, and O-SPI components significantly improved the properties of the gel. Additionally, MP-ASPI gel exhibits the best properties and microstructure. Overall, the four industrial modification effects may impact SPI’s structure and gel properties in different ways. A-SPI could be a potential functionality-enhanced soy protein ingredient in comminuted meat products. The present study results will provide a theoretical basis for the industrialized production of SPI.Native soy protein isolate (N-SPI) has a low denaturation point and low solubility, limiting its industrial application. The influence of different industrial modification methods (heat (H), alkaline (A), glycosylation (G), and oxidation (O)) on the structure of SPI, the properties of the gel, and the gel properties of soy protein isolate (SPI) in myofibril protein (MP) was evaluated. The study found that four industrial modifications did not influence the subunit composition of SPI. However, the four industrial modifications altered SPI’s secondary structure and disulfide bond conformation content. A-SPI exhibits the highest surface hydrophobicity and I Soy protein isolate (SPI) is a vegetable protein with high nutritional value. It is widely used in the food industry due to its balanced composition of basic amino acids, desirable functional characteristics, and low cost . SPI is SPI is frequently used in the food industry because of its ability to form a gel with desirable sensory and physicochemical characteristics. The ability to form gel is the basis for traditional Asian soy products, such as tofu. In addition, SPI is often applied to improve the texture of sausage. Replacing meat protein in food formulations with other sources has attracted growing interest . SPI hasTherefore, evaluating the impacts of SPI structure and the properties of the gel resulting from the four industrial modifications, as well as the gel properties of SPI-MP composites, is essential. This study explains the association between SPI structural changes and gel features and investigates the gel properties of the SPI-MP composite. The findings of this study provide a comprehensive set of data for industrialized modified SPI improvement and can be compared with laboratory data. This work further reveals the functional properties of industrialized modified SPI and the potential of SPI as an additive in meat products, which will provide process parameters and conditions for materials used for producing SPI with high industrial value. It will also provide a theoretical basis for further development and application of soy protein products.Native SPI, industrial heat-modified SPI, industrial alkali-modified SPI, industrial glycosylation-modified SPI, and industrial oxidation-modified SPI were all prepared in Shandong Wonderful Industrial Group Co., Ltd. . In addition, pork longissimus muscle (24 h postmortem) was acquired from a profitable local Walmart store , transported to the laboratory, and used on the same day. All of the substances and components were of analytical degree.The industrialized invention process of SPI was performed according to the assay described by Campbell et al. . As showThe procedures were modified as follows:① H-SPI: The flash temperature in the flash evaporation step was adjusted from 72 °C to 81 °C, and the other parameters remained unchanged.② A-SPI: The pH of the neutralization sample was amended from 7.3 to 8.7, and the other parameters remained unchanged.③ G-SPI: 1% konjac starch was added during formulation, and the mixture was heated and stirred to 75 °C for 90 min. The other parameters remained unchanged.④ O-SPI: The SPI was placed in a 45 °C storage warehouse for one week for oxidation, and the other parameters remained unchanged.Electropherograms of industrially modified SPI on sodium dodecyl sulfate-polyacrylamide gels were obtained using the assay described in Laemmli et al. . Briefly–1 range with a 4.0 cm–1 spectral resolution and 30 s of exposure time. Using Lab spec software, individual band intensities were smoothed, adjusted for baseline, and standardized using the phenylalanine band at 1004 cm−1.Following the procedures of Xiao et al. , with a Using an ANS approach with minor alterations (Kato and Nakai) , the Ho g). We estimated the values using a molar extinction coefficient of 13,600 M−1 cm−1 and a supernatant absorbance at 412 nm.Using Wang’s method  with a f−1. Pyris 6.0 software was used to acquire and process the data, resulting in the DSC curve of SPI. SPI’s denaturation temperature (TD) was obtained from the peak temperature, and the denaturation enthalpy change (ΔH) was computed from the area between the curve and the baseline.Tang’s assay was followed to determine the thermal stability of the SPI structure using a differential scanning calorimeter . Five milligrams of SPI samples with different industrial modification treatments were weighed, blended with 10 μL of 0.01 mol/L phosphate buffer (pH 7.0), placed in an aluminum plate, sealed, and kept at RT for 8 h. The aluminum pan containing the sample was located on the left edge of the DSC instrument console, while the blank aluminum pan was positioned on the right edge. The temperature scanning scale was 20~110 °C, and the heating ratio was 10 °C min2O, magnetically stirred for 4 h at RT, and centrifugated at 10,000× g for 30 min. The protein concentration in the upper phase was measured by the biuret assay, and the protein dose in the supernatant was diluted to 10% (w/w). A rotational rheometer  was used for the measurement after vacuum degassing. The rheometer dish with a width of 40 mm and 0° was selected, the probe size was 20 mm, and the plate spacing was 1 mm. The plates were filled with a protein solution, and the excess solution was wiped off. Small drophosphate buffers of silicone oil were attached to the top of the bowl, and a protective shield was placed to avoid water evaporation.SPI samples were dissolved in HThe initial temperature was 20 °C, then raised to 95 °C at 2 °C/min. The temperature was held at 95 °C for 30 min, then reduced to 20 °C at 2 °C/min. The angular frequency was set to 0.63 rad/s throughout the process, and the maximum strain was 1%. The parameters such as G′ and G″ in the whole process were measured.After the temperature sweep, the temperature was held at 20 °C, the maximum strain was 1%, and the sweep frequency was 0.1~10 Hz. The parameters such as G′ and G″ in the whole process were measured.Gels were created by heating from 20 to 80 °C at a rate of 1 °C/min. Each sample was repeatedly sheared in an oscillatory mode with a maximum strain of 0.02 at a fixed frequency of 0.1 Hz. For the rheological characterization of the gels, the storage modulus (G′) and loss modulus (G″) were quantified.Xia, Kong, Liu, and Liu  used thew/v) using distilled water in a 40 mM phosphate buffer that included 0.6 M NaCl (pH 6.25). MP and SPI were mixed in a 4:1 ratio and homogenized to achieve a 40 mg/mL protein content and adjusted to pH 7.0 in the well-mixed composite protein solution.MP extract with a protein content of about 3% was diluted to 40 mg/mL. SPI  was prepared at 40 mg/mL  for 30 min before each measurement. For convenience, the composite gels prepared from MP with added N-SPI, H-SPI, G-SPI, and O-SPI are abbreviated as MP-NSPI, MP-HSPI, MP-ASPI, MP-GSPI, and MP-OSPI, respectively.A texture profile analysis (TPA) of the SPI-MP gels was performed using a texture analyzer  equipped with probe P/36R (3.6 cm diameter), as described by  with minor modifications. The gel samples were cut into cylinders , equilibrated for 2 h at room temperature, and then compressed at a compression degree of 50%. The pre-test and post-test speeds were set at 3 mm/s, and the test speed was set at 0.3 mm/s to obtain the TPA parameters . The trigger type was set to auto with a 5.0 g trigger force. All samples are measured in 6–8 parallel.w/w) of H2O maintained in the gel after centrifugation , using the following equation:0 and W1 represent the weight (g) of the gel before centrifugation and the weight (g) of the gel after centrifugation, respectively.The WHC (water holding capacity) (%) of MP gel was determined via the assay explained by Niu, et al. . The fin3) created via the technique above were placed, for at least 1.5 h, in a 2.5% glutaraldehyde 0.1 M phosphate buffer (pH 7.0). Then, after being washed three times with the same buffer for 10 min, the gel samples were dehydrated by being dipped in a series of solutions  of ethanol for 10 min per solution. Finally, each sample was lyophilized, sputter-coated with gold, and observed at an acceleration voltage of 10 kV and a magnification of 1000. In the SEM observations, there were three replications. For each replica, six pictures were taken.The process described by Haga and Ohashi  was modiThe results were provided as mean ± SD for every experiment conducted at least in triplicate. Data were processed using one-way ANOVA and Duncan’s test in the SPSS statistical package . The statistical significance between the two means was determined at the 95% confidence level.SDS-PAGE was used to monitor subunit alterations in SPI after the industrial modification treatments. As shown in −1), the tyrosine double chain (I850/I830), and the amide I band (1600–1700 cm−1). Spectral data were evaluated in 3 wavelength zones  to study protein structure changes during processing, focusing on disulfide bond conformation, tyrosine environment, and secondary structural changes.−1 indicate alterations in protein secondary structure. The characteristic peaks at 1652, 1663, 1674, and 1683 cm−1 correspond to α-helix, random coil, β-sheet, and β-turn, respectively [Changes in the amide I band between 1600 and 1700 cmectively . We fittectively  found thIn contrast, β-turns and random coils represent semi-regular arrangements . IndustrLi et al. reported that greater electrostatic revulsion among protein fragments might be responsible for reducing the content of irregular coils . Industr−1 zone. The creation of different disulfide bond conformations is implicated in the S-S conformational changes. Disulfide bonds can affect protein aggregation and gel properties, as they play a potential role in protein aggregation dissemination and gel creation [−1), gauche-gauche-trans-S-S  and ‘trans-gauche-trans-S-S . T-g-t denotes inter-chain disulfide connections among proteins, whereas g-g-g and g-g-t modes reflect intrachain disulfide bonds (S-S) inside proteins [Table 1, compared to the control, the g-g-g configuration of A-SPI was not significantly adjusted (p > 0.05), the relative quantity of the g-g-t conformation declined, and the comparative quantity of the t-g-t conformation was heightened. This may be because the disulfide bonds among the protein molecules (g-g-t) break easily in alkaline environments [The impact of various industrial alteration treatments on the S-S conformational changes of SPI was assessed by displaying S-S stretching vibrations in the 500–550 cmcreation . The majproteins . A possiproteins . As showronments  and becaronments . Additioronments . In the ronments .−1 (I850/830) displays polarity and hydrogen bond formation in the microenvironment surrounding the phenolic hydroxyl groups. A decline in the I850/830 proportion reflects a rise in the burial of Tyr residues, and an increase in the I850/I830 ratio reflects a rise in the disclosure of Tyr remains and the forming of hydrogen bonds [850/I830 results for different industrially modified SPI samples are shown in 850/I830 ratio (p > 0.05), probably because industrial heat treatment enhanced the tendency of Tyr residues to act as hydrogen bond donors to augment internal hydrogen bonding, but did not significantly alter the polarity of the tyrosine residue microenvironment [2O, increases the I850/I830 ratio.The ratio of the bimodal tyrosine band between 850 and 830 cmen bonds . The I85ironment . Industrironment . Li et aironment  showed tironment . This maironment . The incSurface hydrophobicity characterizes the degree of exposure of internal hydrophobic groups and gelation characteristics. Hydrophobicity is the main force during gel formation , affectiThe creation of aggregates in proteins is primarily attributed to disulfide bonds. Sulfhydryl groups (-SH) can affect disulfide bond formation through SH-SS exchange reactions, regulating protein aggregation and gelation . Total sSome active –SH groups interact with cysteine-containing residues through S-S bonds, forming disulfide bonds. This process results in a decrease in the quantity of active –SH groups. However, the unfolding of the spatial structure exposes internal SH groups, leading to an increase in the quantity of active –SH groups . This maD) of the proteins as they were continuously heated to disrupt their advanced structures. The DSC curves for the different industrially modified SPI are presented in D values indicated the changes in thermal stability, while the changes in ∆H suggested the flexibility of the protein structure and the ease of denaturation [D and no significant change in 11S TD in H-SPI compared to N-SPI.The functional features of proteins could be compromised by heat treatment, resulting in protein denaturation or aggregation. In this study, differential scanning calorimetry (DSC) was employed to evaluate the degree of denaturation of different industrially modified SPI during heat treatment by measuring the energy alteration (∆H) and denaturation temperature  of the complex. Additionally, introducing sugar molecules may break covalent or non-covalent bonds between proteins during glycosylation, causing the protein structure to become looser. As a result, the 7s and 11s subunits in G-SPI are more susceptible to unfolding during continuous heating, and the ΔH of the 7s and 11s subunits in G-SPI is reduced [The industrial glycosylation treatment process leads to the binding of SPI and konjac flour starch molecules, causing polysaccharide molecules to affect SPI molecules in space, which, to some extent, prevents the thermal aggregation of proteins. The resulting glycoprotein can maintain a more stable conformation when heated, improving the thermal stability of SPI and increasing the denaturation temperature  of different industrially modified SPI and MP composite gels was evaluated. The water retention results of the MP-SPI composite gel and the MP gel are shown in ving WHC .The rheological features of the MP-SPI composite gel system were investigated by determining the shift in the storage modulus (G′) through heating. G′ represents the energy stored in the gel sample through heating and is an essential dynamic rheological indicator that reflects its elastic actions . MP undeA final temperature of 65–74 °C was satisfactory for general meat processing circumstances. Nevertheless, this temperature was inadequate to denature 100% of the N-SPI protein structure. This may clarify why N-SPI did not contribute to the gel properties of MP-NSPI. N-SPI has a tightly packed spherical structure that prevents interaction with meat proteins, resulting in reduced cross-links among protein aggregates . This maThe functional features of gels, such as WHC, strength, and rheology, are determined by their three-dimensional networks. The physical features of the gel structures are closely related to their microstructures. The structures of composite gels made from SPI and MP after various industrial modifications were observed using scanning electron microscopy (SEM) (×1000) . AssociaIn addition, more hydrophobic residues (highest Ho, This study examined the impacts of four industrial modifications on the gel properties of SPI and the structure of SPI, as well as its complexation with MP in the preparation of protein gels. None of the industrial modifications significantly influenced the subunit composition of SPI. However, they increased the content of β-fold and β-turned structures and decreased thermal stability. All of the modifications decreased the gelling temperature point and G′ of SPI, resulting in faster protein aggregation. The gel intensity, WHC, and texture features of the MP-SPI composite gels were significantly enhanced by the modifications, particularly the alkali modification treatment. The addition of N-SPI disrupted myosin interactions and adversely affected the formation of MP gels. The four industrial modification treatments can be applied to SPI to improve its gelation ability. We observed that MP-ASPI had the highest gel strength and the most continuous, dense gel network structure. Therefore, a-SPI could be used as a potential functionality-enhanced soy protein ingredient in comminuted meat products. The research results will guide food companies to develop industrial modification processes based on laboratory test results, provide high-quality raw material production methods for producing high-value SPI products, and have important practical significance for efficiently using the variety of soybean resources in the future market economy.


Improved survival of cancer patients leads to more skeletal metastatic lesions that need local therapies for tumor control and pain relief. Not all tumors are radiosensitive and alternative therapies are direly needed. Microwave ablation (MWA) is a technique for minimally invasive local tumor control by physical ablation. In soft tissue local temperature ablation is more common, but studies on bone tissue are limited. To ensure safe and effective treatment, studies on local tumor ablation in bone are needed.Microwave ablation was performed on sheep bone, for both in- and ex-vivo settings. Both a slow-cooking MWA protocol  and a fast-cooking protocol (no warm-up period) were used. Heat distribution through the bone during ablation was determined by measuring temperature at 10- and 15mm from the ablation probe (= needle). Ablation size after procedure was measured using nitro-BT staining.In-vivo ablations led to up to six times larger halos than ex-vivo with the same settings. Within both ex- and in-vivo experiments, no differences in halo size or temperature were found for different wattage levels (65W vs 80W). Compared to a fast cooking protocol, a two-minute slow cooking protocol led to increased temperatures and larger halos. Temperatures at 10- and 15mm distance from the needle no longer increased after six minutes. Halo sizes kept increasing over time without an evident plateau.Microwave ablation is technically effective for creating cell death in (sheep) long bone. It is recommended to start ablations with a slow-cooking period, gradually increasing the surrounding tissue temperature in two minutes from 40 to 90°C. Ex-vivo results cannot simply be translated to in-vivo. Improved survival of cancer patients leads to more skeletal metastatic lesions that are amenable to local therapies for tumor control and pain relief . Not all°C in the complete tumor. It is expected that temperature decreases during conduction through the tissue as a result of (micro)vascularity. After ablation with temperatures under 60°C tissue has the capability to recover, leading to possible tumor recurrence. At temperatures over 100°C vaporization of water (leading to desiccation of the cell) and carbonization occur. These factors potentially limit heat conduction [At temperatures over 60°C instant cell death occurs by coagulative necrosis. Between 50–60°C it takes 1–6 minutes to reach complete cell death . Therefonduction –14. In Fnduction .Literature on safety and efficacy of local temperature ablation in bone tissue is still limited. A wide range of wattage levels is used for clinical MWA. Given the fact MWA leads to larger ablation zones compared to alternatives like radiofrequency ablation, clinical use might result in excessive margin or incomplete ablation. An excessive margin could lead to damage to surrounding structures, incomplete ablation to lack of local control . To ensuIn this study we compared different settings to study the effects of wattage level and ablation time on halo size.All ablations were performed with a 2.45GHz Kang-You 2000 microwave ablation generator . The needle had an active part of three cm long and a diameter of 17 gauge. Internal cooling of the needle was performed, preventing temperatures over 45°C in the non-active part. Needles were cleaned with alcohol between procedures and replaced when the protective insulation layer started to show damage. An ex-vivo study was performed to determine heat distribution and halo size for a wide range of settings. Subsequently the most promising settings were transferred into an in-vivo study.Fresh sheep femur (from sheep aged 1–1.5 years with an average weight of 50kg) were collected from the slaughterhouse. These bones were chosen given the similarity to human bone anatomy. The lower extremity is the most common location for metastases and tumors in human bone . AblatioTo prevent carbonization as a result of over-rapid temperature increase around the needle tip (which is thought to limit heat distribution), a protocol for slow-cooking was developed. According to this protocol wattage was slowly increased towards the aimed setting: 10W for one minute, followed by one minute at 20W. These two minutes were included in the time intervals as mentioned for the slow-cooking ablations (see next paragraph). Furthermore, a fast-cooking protocol was developed. In this protocol ablation was directly started at the aimed maximum wattage. Results of both protocols were compared.The tract for the ablation needle was drilled in the proximal metaphysis of sheep femur at a depth of 30mm. Through this tract the ablation needle was brought up. Temperature was measured by a Kang-You temperature probe (21 gauge). Tracts for the temperature probe were drilled 5, 10 or 15mm lower in the same direction as the ablation needle see . Only onA total of ten settings was used, with wattages of 65W  and 80W . The aim of this was to determine the contribution of each parameter or setting on halo size. For each setting 10 measurements were performed.20, one part phosphate buffer solution and one part Nitro-BT [Between two and eight hours after ablation the bones were cut in the coronal plane at the insertion point of the needle using an automatic saw with a 1-mm thick blade. Subsequently the coupes were stained in a Nitro-BT solution (nitro blue tetrazolium chloride) and incubated in a 37°C incubator for three hours see . This soNitro-BT . As a reIn-vivo MWA was performed in 12 sheep femurs, using the same protocol as the ex-vivo experiments. For four sheep ablation was performed with 65W for six minutes, for four sheep 65W for 12 minutes and for four sheep 80W for six minutes. Heat distribution was measured at 10- and 15mm for 65W ablations (one distance per ablation) and at 10mm for 80W ablations (not at 15mm based on ex-vivo results and to limit the number of sheep needed). Before starting the procedure, the sheep were sedated by a veterinarian with an intramuscular injection of 5 ml Shutai (1: 1 combination of Tiletamine 50mg/ml & Zolazepam hydrochloride 50mg/ml)  and subsequently intubated. During the procedure inhaled isoflurane (1–5%) was used for general anesthesia and a maintenance dose up to 2.5ml Shutai was given in case deemed necessary by the veterinarian. Sheep were sacrificed one week after the procedure, bones were cut and halo size was determined after staining (with the same procedure as ex-vivo). The in-vivo study was approved by the medical ethical committee of the Xi’an HongHui hospital.Technical issues like sudden temperature drops, temperature differences more than two standard deviations (SDs) from the average value  and high impedance (dessicated tissue in a few ex-vivo bones) warranted exclusion from the analysis.Data were analyzed using SPSS v25 . Values for temperature and halo dimensions were noted as mean (±SD). Halo volume was calculated according to the formula: 4/3*pi*(1/2*height)*(1/2*width)*(1/2*depth) = 1/6*pi*height*width*depth. Data were examined using non-parametric tests due to the relatively small group size. Differences between fast- and slow-cooking, 65W and 80W, and in- and ex-vivo were tested using a mixed repeated measures ANOVA design. Sphericity and equality of differences were controlled. Data were tested for time up to six minutes . For all tests a p-value of < .05 was considered significant.For the slow-cooking samples, temperature remained below 60°C during the two-minute slow-cooking period. For both 65W and 80W temperature exceeded 60°C within three minutes at 10mm and within five minutes at 15mm. There was no significant difference in temperature over time between 65W and 80W  = 1.43, p = .228, at 15mm F = 1.68, p = .152). For the fast-cooking samples, initial temperature increase was fast with temperature reaching up to 60°C within two minutes at 10mm from the needle. At 15mm temperature increase was similar to the slow-cooking samples. After the initial fast increase, temperatures increased more gradually compared to the slow-cooking samples. From five minutes onwards temperature was significantly lower at both 10mm  = 35.19, p < .01) and 15mm  = 7.23, p < .01). The values are depicted in Halo volume gradually increased over time. The major part of the halo was formed within the first six minutes. Halo size continued to increase up to the 12-minute measurement but was not significantly larger at twelve minutes compared to six minutes for the slow cooking samples . For the fast cooking samples halo size after 12 minutes was significantly larger (p = .024). This is contrary to the heat distribution data where almost no further increase was seen in the period after six minutes. There was no difference in halo size between 65W and 80W (p = .890) or between fast- and slow-cooking (p = .861). Values for volume are depicted in For in-vivo ablations, heat distribution was significantly smaller compared to ex-vivo  = 17.69, p < .01, at 15mm F = 6.58, p < .01). At 10mm temperature reached over 60°C after four to five minutes (for 80W and 65W respectively), at 15mm (with 65W) this occurred after nine minutes. Temperature remained below the ‘safe’ 60°C for a longer time than in ex-vivo. Values are depicted in Halo size in-vivo was significantly larger than ex-vivo (p < .01). Halo size did not significantly increase between six and twelve minutes ablation (p = .857). There was no significant difference in halo size between 65W and 80W either (p = .057). This study is the first to examine the effect of different settings for microwave ablation in long bone in an experimental setting. Since both ex-vivo and in-vivo experiments were performed, results are likely transferrable to the clinical setting. Results prove microwave ablation is technically effective for creating cell death in (sheep) long bone in a minimally invasive manner. Ablation with 65W is sufficient, the major portion of cell death is formed in the first six minutes of ablation, slow-cooking is better than fast-cooking and ex-vivo halo size is smaller than in-vivo halo size. This data indicates ‘aggressive’ ablation with high wattages and longer time intervals does not lead to better results. To reduce risk of complications ablation settings must be determined with caution.Assumptions regarding the relationship between cell death and temperature as postulated in literature see  do not mTemperatures at 10- and 15mm from the needle were higher ex-vivo than in vivo. This is most likely due to in-vivo heat sink, for instance by (micro)vascularity in and around the bone. In the study of Ji et al. they measured temperature at 5, 10, 15 and 20mm  from the needle tip during a 36-minute microwave ablation in dog femur (amount of Watt not described) . They foInterestingly, halo size was larger in the in-vivo samples than ex-vivo, regardless of the lower temperatures at 10- and 15mm from the needle. A possible explanation for the difference between in- and ex-vivo halo size is the time between ablation and halo measurement. In the in-vivo experiment the animals were sacrificed after one week and subsequently halo size was measured whereas ex-vivo measurement was done within hours after the ablation. The cells that were identified dead in the in-vivo experiment could be the effect of both necrosis as well as apoptosis, whereas in the ex-vivo experiment only the effect of necrosis can be seen . There might also be an effect of late-apoptosis in the in-vivo experiments, for instance because of ROS activity. However, it is not likely that differences as large as found in our data only originate from follow-up time after ablation ,21.An increase in wattage level from 65W to 80W did not lead to larger halos or higher temperatures at 10- and 15mm from the needle. Therefore, a wattage of 65W is considered sufficient. Further increase of the wattage only increases risk of complications. Potentially a similar outcome could be found for even lower wattage levels. This could be examined in further research. Between fast- and slow cooking significant differences in heat distribution were seen. In the fast-cooking group temperature at 10- and 15mm is higher during the first two minutes of ablation compared to slow-cooking. However, from five minutes onwards the temperature of the slow-cooking group is significantly higher. This effect is hypothesized to be the result of carbonization around the needle as a result of over-rapid heating in the fast-cooking samples. This carbonized tissue can  block the distribution of heat through the bone. Therefore, we recommend to use a two-minute slow-cooking period at every ablation to ensure gradual heating of the tissue. Since halo size mainly increased in the first six minutes, longer ablations than six minutes are not encouraged.This study had some limitations. First, positioning of the drill was done by hand and without image-guidance. Therefore, small differences between the measurements cannot be ruled out. For all ex-vivo experiments at least seven samples were included to ensure a reliable outcome. Distance between needle and temperature probe was standardized using a navigation tool, thereby also ensuring same direction and limiting variation between samples as a result of human error. This tool was also used for the in-vivo experiments. Second, for the in vivo experiments only four animals were used per setting and there was substantial variance in halo size between the animals. For the in-vivo study animals were sacrificed after one week and subsequently analyzed whereas ex-vivo analysis was performed on the day of ablation. Finally, experiments were performed in healthy bone without tumor tissue.Microwave ablation is technically effective for creating cell death in (sheep) long bone. It is recommended to start ablations with a slow-cooking period, gradually increasing the surrounding tissue temperature in two minutes from 40 to 90°C. Ex-vivo results cannot simply be translated to in-vivo, given in-vivo halo volume was up to six times larger than ex-vivo. The technique still faces issues like high variability and therefore limited predictability, before it can become mainstream treatment. Future experiments and development should focus on ways to reduce this.S1 Data set(XLSX)Click here for additional data file.